Regulation by-anti-sense RNA | Regulation of Gene Expression Operon Circuits in Bacteria and other Prokaryotes | Genetics, Biotechnology, Molecular Biology, Botany

⇒	Regulation of Gene Expression 1. Operon Circuits in Bacteria and other Prokaryotes
⇒	Induction and repression
⇒	Inducer and co-repressor
⇒	The operon model for transcriptional regulation
	⇒	The tryptophan operon in bacteria (E. coli and Salmonella)
	⇒	Tryptophan (trp) repressor controls three sets of genes
	⇒	Negative and Positive Controls of Transcription
	⇒	Substitution of Sigma Factor and Control of Transcription
	⇒	Multiple sigma factors in E. coli
	⇒	Sporulation in bacteria
⇒	DNA sequences controlling transcription
	⇒	DNA sequences for CAP, RNA polymerase and lac-repressor
	⇒	Identification of starting point
	⇒	Pribnow box and other sequences common to DNA regions upstream to several operons
⇒	Regulation by DNA rearrangements
⇒	Post-transcriptional regulation
	⇒	Leader sequences and attenuators
	⇒	Autogenous regulation of translation
	⇒	Regulation by alternative splicing
	⇒	Regulation by-anti-sense RNA
	⇒	Repression and activation of translation
	⇒	Feedback inhibition
⇒	Signal transduction and ‘two component regulatory system’

Regulation by-anti-sense RNA
During late 1980's and early 1990's, it has been shown that the translational control of protein synthesis can be exercised by using RNA, which is complementary to mRNA, so that this introduced RNA may form RNA-mRNA hybrids and prevents mRNA from being translated. Since this RNA will be complementary to mRNA and will interfere with its translation, it is called anti-sense RNA or mic RNA (mic = mRNA interfering complementary RNA). Such anti-sense RNA can be used to stop translation of mRNA in cells at will, and therefore is reported as a method analogous to production of mutations at will. But more important is the use of this phenomenon by cells to exercise control of gene expression at the level of translation.

In transposon Tn10, translation leading to the production of transposase (a protein required for transposition) is inhibited through the production of anti-sense RNA. The production of anti-sense RNA is controlled by a promoter which directs transcription in opposite direction to the transposase gene, producing anti-sense RNA, which overlaps 36 bases of mRNA at its 5' end, and are complementary to it. There is definite evidence that the synthesis of transposase enzyme in Tn10 is regulated through the synthesis of anti-sense RNA.

In E. coli also, the role of anti-sense RNA in regulating gene expression has been demonstrated in case of two genes for outer membrane protein. These two genes are ompC and ompF (Omp = outer membrane protein). The total amount of these two proteins remains constant, although their relative quantities may differ according to requirement. It has been shown that a DNA fragment upstream of ompC when introduced into omp F⁺ cells, inhibited the production of OmpF protein. This DNA fragment coded for a small 174 base RNA and is termed mic RNA (mRNA interfering complementary RNA) which was transcribed in a direction opposite to ompC gene. Since no ribosome binding site is present on mic RNA, it can not be used for protein synthesis. But it has extensive homology with 5' end of ompF mRNA, and therefore, can form a hybrid with it. Since the sequences present in mic RNA include sequences complementary to ribosome binding site of ompF mRNA, hybrid formation will inhibit the translation initiation. Such a mechanism is believed to result in coordinated regulation of the production of OmpC and OmpF proteins.

Even in eukaryotes, it is suggested that anti-sense RNA (or even anti-sense DNA) may be used to inhibit gene expression and the presence of complementary RNA (or DNA) sequence was shown to reduce gene expression. The extent of utilization of this mechanism for control of gene expression in cell is not known, but it is being definitely utilized now for artificial manipulation of gene expression.

Inhibition of the expression of cloned genes by antisense RNA/DNA technology has been achieved using recombinant-DNA technology through the construction of antisense expression vectors (for details consult Genetic Engineering and Biotechnology 1. Recombinant DNA and PCR (Cloning and Amplification of DNA)). These genes whose expression was thus manipulated, included the following : (i) thymidine kinase (TK) gene from herpes simplex virus (HSV), (ii) actin gene (actin protein is a major component of cytoskeleton), (iii) genes for pigment synthesizing enzyme in petunias; (inhibition of this enzyme led to unusual pigmentation in flowers), (iv) genes for enzymes responsible for fruit ripening in tomatoes (inhibition of these genes will help in slow ripening) and (v) plant virus genes causing diseases. In all these cases, an antisense expression vector was constructed, so that the orientation of the gene of interest is reversed with reference to promoter sequence. These will lead to the synthesis of antisense RNA inhibiting the translation of mRNA. Using this technology inhibition of the expression of several genes as above was achieved successfully.

Not only antisense RNA, but artificially synthesized oligonucleotides (both DNA and RNA) have also been used to inhibit the translation of mRNA. This technology will be increasingly used in future for study of gene function and for treatment of diseases.





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